Promiscuous PAP CD4 T cell epitopes

ABSTRACT

The present invention relates to the discovery of novel T cell epitopes of the human prostatic acid phosphatase (PAP) protein that is promiscuous for at least 15 different HLA-DR alleles. The invention also relates to compositions that contain one of the novel epitopes or a fusion peptide of such an epitope and a heterologous polypeptide. Further disclosed herein is the use of the epitopes or their fusion peptides, and compositions containing the epitopes or their fusion peptides.

RELATED APPLICATIONS

This application claims priority to provisional U.S. Patent ApplicationNo. 60/837,053, filed Aug. 11, 2006, the contents of which areincorporated herein by reference in the entirety.

BACKGROUND OF THE INVENTION

HLA class II-restricted CD4+ T cells play a critical role in cellularimmunity and are a key component of anti-tumor immune responses. CD4+ Tcells provide necessary help to tumor-specific CTLs (Topalian 1994. CurrOpin Immunol 6:741-745) and produce cytokines such as interferon gamma(IFNγ), which can activate antigen presenting cells and mediate otherimmunological effects (Corthay et al., 2005. Immunity 22:371-383).Experimental results in several systems have demonstrated that CD4+ Tcells are necessary for an effective anti-tumor immune response. Giventhe importance of CD4+ T cells in generating a robust immune response,an optimally designed cancer immunotherapy or anti-tumor vaccine shouldinduce both tumor-specific CD4+ and CD8+ T cells for maximal efficacy.

The human prostatic acid phosphatase (PAP) is a phosphatasepredominantly expressed in the prostate gland. Elevated serum level ofPAP is often observed in patients with prostate cancer or other prostateconditions, with the highest levels of PAP found in metastasizedprostate cancer. In addition, diseases of the bone, such as Paget'sdisease or hyperparathyroidism, diseases of blood cells, such assickle-cell disease, multiple myeloma, or lysosomal storage diseases,such as Gaucher's disease, will show moderately increased levels of PAP.Because over 95% of prostate cancer cells express PAP, severalimmunotherapeutic strategies for prostate cancer have been devised usingPAP as a target. For instance, Cancer Biology and Therapy (March 2005,vol. 4, issue 3) has reported promising results from a clinical study inwhich a patient's own immune cells were collected, stimulated to becomeimmunoreactive to PAP, and then returned to the patient by intravenousinjection. These new immunological approaches rely on methods that caneffectively induce a PAP-specific immunity, including T cell-mediatedimmunity.

The usefulness of a defined T cell epitope is limited by itsHLA-restriction. Peptide epitopes typically form productive peptide-MHCcomplexes with a small number of HLA alleles and stimulate T cellresponses only in individuals expressing those alleles. This confinesimmunological studies and clinical trials to individuals of a specificHLA type, often 20% or less of the general population. So-calledpromiscuous T cell epitopes, which can be presented by a larger numberof HLA alleles, have been described for several tumor antigens.Promiscuous T cell epitopes can bind to multiple HLA alleles tostimulate antigen-specific T cells, allowing for the induction and studyof T cell responses in individuals of different HLA types. Additionally,promiscuous epitopes are valuable because the immunotherapies andvaccines based on these epitopes can be widely applicable to the generalpopulation for cancer treatment and prevention. Thus, there exists aclear need for new information relating to previously unknownpromiscuous epitopes of tumor antigens, including PAP.

The present inventors have identified a series of novel promiscuous Tcell epitopes in the human PAP protein sequence. These epitopes,comprising the region of 257-271 of the PAP protein, are recognized by aCD4+ T cell clone (PAPc66) in the context of HLA-DR and capable ofinducing T cell activation when presented by antigen presenting cell ofat least 15 different HLA-DRβ1* alleles. The promiscuity of theseepitopes for different HLA-DRβ1* alleles makes these epitopes a valuabletool for evaluating PAP-specific immune responses regardless of thepatient's HLA type. Additionally, these epitopes can be used as auniversal CD4 T helper cell epitope in peptide-based vaccines orimmunotherapies for the treatment PAP+ prostate cancers.

BRIEF SUMMARY OF THE INVENTION

The present invention describes novel PAP epitopes that can be presentedby antigen presenting cells of a number of different HLA alleles toinduce a PAP specific T cell response. In the first aspect, thisinvention provides an isolated peptide of 15-18 amino acids, whichcomprises a segment derived from the human PAP protein sequence(residues 257-271, i.e., SEQ ID NO:1). Preferably, the peptide consistsof 15 to 18 contiguous amino acids of SEQ ID NO:2, which corresponds tothe 254-274 segment of human PAP protein sequence. More preferably, thepeptide has the sequence set forth in SEQ ID NO: 1. Also provided is afusion product comprising the PAP derived peptide fused to aheterologous polypeptide. In some cases, the peptide is fused to theheterologous polypeptide by a peptide bond, such that the fusion productis in essence a recombinant fusion protein.

Preferably, the isolated peptide derived from the PAP sequence or itsfusion product is capable of inducing a T cell immune response specificto a PAP protein when presented by an antigen-presenting cell of atleast 10 different HLA-DR alleles, and more preferably, at least 11, 12,13, 14, 15, or more different HLA-DR alleles.

In some embodiments, the HLA-DR alleles are selected from the groupconsisting of 0101, 0102, 0103, 1503, 160201, 0301, 0302, 0401, 0402,040301, 040501, 1101, 1102, 1103, 1104, 110401, 1201, 1301, 1302, 1401,1402, 0701, 080101, 080201, and 0901.

In some embodiments, the PAP derived peptide has the amino acid sequenceof SEQ ID NO: 1. In other embodiments, the heterologous polypeptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF).

In a second aspect, the present invention provides an isolated nucleicacid comprising a polynucleotide sequence encoding a PAP derived peptidedescribed above or a fusion protein joining a PAP derived peptide and aheterologous polypeptide by a peptide bond, an expression cassettecomprising the nucleic acid, and a host cell comprising the expressioncassette.

In some cases, the polynucleotide sequence encodes the peptide havingthe amino acid sequence of SEQ ID NO: 1. In other cases, thepolynucleotide sequence encodes a fusion protein in which theheterologous polypeptide is GM-CSF.

In some embodiments, the expression cassette is a recombinant viralvector. In other embodiments, the expression cassette directs theexpression of the peptide having the amino acid sequence of SEQ ID NO: 1or a recombinant fusion protein in which the heterologous polypeptide isGM-CSF.

In a third aspect, the present invention provides a compositioncomprising a PAP derived peptide as described above or a fusion productof the peptide fused with a heterologous polypeptide, in addition to aphysiologically acceptable excipient.

In some embodiments, the peptide has the amino acid sequence of SEQ IDNO: 1. In other embodiments, the heterologous polypeptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF). In yet otherembodiments, the composition further comprises an antigen-presentingcell, which has the PAP derived peptide forming a complex with a majorhistocompatibility complex (MHC) molecule on the surface of the cell.

In a fourth aspect, the present invention provides a method for inducingin a patient a T cell immune response specific to a PAP protein. Thismethod comprises the step of administering to the patient an effectiveamount of the composition comprising a PAP derived peptide as describedabove or a fusion product of the peptide fused with a heterologouspolypeptide, as well as a physiologically acceptable excipient.

In some embodiments, the peptide has the amino acid sequence of SEQ IDNO: 1. In other embodiments, the heterologous polypeptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF).

In a fifth aspect, the present invention provides a method for detectingin a patient a T cell immune response specific to a PAP protein. Thismethod comprises the following steps: (a) obtaining anantigen-presenting cell and a T cell from the patient; (b) contactingthe antigen-presenting cell and the T cell with a PAP derived peptide ora fusion product comprising the peptide and a heterologous polypeptide;and (c) detecting a T cell response, wherein the detection of a T cellresponse indicates the presence of a T cell immune response specific toa PAP protein in the patient.

In some embodiments, step (c) is performed by ELISPOT, proliferationassay, or flow cytometry. In other embodiments, the PAP derived peptidehas the amino acid sequence of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. T cell clone PAPc66 recognizes a naturally processed epitopefrom PAP. (A) PAP fusion proteins PA2024, CHOPA2024 and hPAPGM and PAPfrom a baculoviral expression system (iPAP) and mammalian expressionsystem (CHOPAP) were titrated with autologous EBV-LcL and PAPc66. Assaywas set up in 96 well round bottom plates with 1×10⁵ cells/well ofPAPc66, 2×10⁵ cells/well EBV-LcL and antigens at 50 μg/mL (black bars),25 μg/mL (stripe bars) and 12.5 μg/mL (dot bars) in complete RPMI mediawith 10% FBS. Assay was incubated for 48 hours at 37° C. with 5% CO₂ atwhich time supernatants were harvested and tested for IFNγ by ELISA. (B)To map the specific PAP peptide, chemically synthesized peptides fromthe PAP sequence were used. These 94 peptides were 15 amino acids inlength overlapping by 11 amino acids and were individually tested forthe ability to stimulate PAPc66 using autologous EBV-LcL and eachpeptide at 2 μg/mL in the assay. The assay was set up as in (A) andsupernatants were tested for IFNγ by ELISA. (C) Peptide #65, PAP₂₅₇₋₂₇₁,was titrated with autologous EBV-LcL and PAPc66 in an assay set up as in(A). Supernatants were analyzed for peptide specific IFNγ production byELISA.

FIG. 2. PAPc66₁ is MHC class II restricted. PAPc66 was stimulated withautologous EBV-LcL at 2×10⁵ cells/mL and PAP₂₅₇₋₂₇₁ at 2 μg/mL in thepresence of MHC Class II blocking antibodies. The assay was set up in 96well round bottom plates in complete RPMI media+10% FBS with anti-HLA-DR♦ (Dendreon) and anti-HLA-DP ▪ or anti HLA-DQ ▴ (Leinco Technologies,Inc., St. Louis, Mich.) at the concentrations shown. Anti-HLA-A2antibody (∘) was used as a negative control. PAPc66 was added to allwells at 1×10⁵ cells/well and the assay was incubated for 48 hours at37° C. and 5% CO₂. After the incubation, supernatants were harvested andtested for IFNγ by ELISA. IFNγ was not detected for conditions withoutpeptide. These results indicate that PAP₂₅₇₋₂₇₁ is presented both in thecontext of HLA-DR and HLA-DQ.

FIG. 3. PAP₂₅₇₋₂₇₁ is a promiscuous MHC class II PAP epitope recognizedby the T cell clone PAPc66. (A) The peptide, PAP₂₅₇₋₂₇₁(RLQGGVLVNEILNHM;SEQ ID NO:1), recognized by a CD4+ T-cell clone (PAPc66) in the contextof MHC class II, is capable of inducing T-cell activation when presentedby lymphoblastoid cell lines representing 9 different HLA-DR serologicalfamilies (β chain; DR1, DR4, DR7, DRB, DR11, DR12, DR13, DR14, DR15)having over 15 distinct HLA-DRβ1 alleles. As shown in FIG. 2, thepeptide can be presented in the context of HLA-DR or HLA-DQ. The assaywas set up in 96 well round bottom plates with PAPc66 at 1×10⁵cells/well, PAP₂₅₇-271 at 2 μg/mL and 2×10⁵ EBV-LcL per well in completeRPMI+10% FBS. Each HLA-DREBV-LcL line, homozygous for the HLA-DR131allele shown, was tested for the ability to present PAP₂₅₇-271 toPAPc66. An irrelevant PAP peptide and no peptide control were alsoincluded. After 48-hour incubation at 37° C. and 5% CO₂, supernatantswere tested for IFNγ and granzyme B (B) by ELISA. Results are shown withbackground (no peptide) for each EBV-LcL line and PAPc66 subtracted.

DEFINITIONS

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

In this application, the term “amino acid” refers to naturally occurringand synthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure different from the generalchemical structure of an amino acid, but capable of functioning in amanner similar to a naturally occurring amino acid.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, or mRNA encoded by a gene.

When the relative locations of elements in a polynucleotide sequence areconcerned, a “downstream” location is one at the 3′ side of a referencepoint, and an “upstream” location is one at the 5′ side of a referencepoint.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds. In this application, the aminoacid sequence of a polypeptide is presented from the N-terminus to theC-terminus. In other words, when describing an amino acid sequence of apeptide, the first amino acid from the N-terminus is referred to as the“first amino acid.”

When used in the context of describing partners of a fusion peptide, theterm “heterologous” refers to the relationship of one peptide fusionpartner to the another peptide fusion partner: the manner in which thefusion partners are present in the fusion peptide is not one that can befound a naturally occurring protein. For instance, a “heterologouspolypeptide” fused with a promiscuous prostatic acid phosphatase (PAP)epitope to form a fusion peptide may be one that is originated from aprotein other than the PAP protein, such as a granulocyte-macrophagecolony-stimulating factor (GM-CSF). On the other hand, a “heterologouspolypeptide” may be one derived from another portion of the PAP proteinthat is not immediately contiguous to the promiscuous epitope. A“heterologous polypeptide” may contain modifications of a naturallyoccurring protein sequence or a portion thereof, such as deletions,additions, or substitutions of one or more amino acid residues.Regardless of the origin of the “heterologous polypeptide” (i.e.,whether it is derived from the PAP protein or another protein), thefusion peptide should not contain a subsequence of the PAP protein thatencompasses the amino acid sequence of SEQ ID NO: 1 and have more than18 amino acids in length. In some exemplary embodiments, a “heterologouspolypeptide” for use in the present invention has no more than 15-20amino acids in length; in other embodiments, a “heterologouspolypeptide” has at least 100 amino acids in length.

The word “fuse” or “fused,” as used in the context of describing apeptide of this invention that comprises a promiscuous PAP epitopejoined with a heterologous polypeptide, refers to a connection betweenthe epitope and the heterologous polypeptide by any covalent bond,including a peptide bond.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences that may be introduced to conform to codon preference in aspecific host cell.

An “expression cassette” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular polynucleotidesequence in a host cell. An expression cassette may be part of aplasmid, viral genome, or nucleic acid fragment. Typically, anexpression cassette includes a polynucleotide to be transcribed,operably linked to a promoter.

The term “recombinant,” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of a nucleicacid or protein from an outside source or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all.

The term “administration” or “administering” refers to various methodsof contacting a substance with a mammal, especially a human. Modes ofadministration may include, but are not limited to, methods that involvecontacting the substance intravenously, intraperitoneally, intranasally,transdermally, topically, subcutaneously, parentally, intramuscularly,orally, or systemically, and via injection, ingestion, inhalation,implantation, or adsorption by any other means. One exemplary means ofadministration of the promiscuous PAP peptide of this invention or afusion peptide comprising the PAP peptide and a heterologous polypeptideis via intravenous delivery, where the peptide or fusion peptide can beformulated as a pharmaceutical composition in the form suitable forintravenous injection, such as an aqueous solution, a suspension, or anemulsion, etc. Other means for delivering the promiscuous PAP peptide ora fusion peptide of this invention includes intradermal injection,subcutaneous injection, intramuscular injection or transdermalapplication as with a patch.

An “effective amount” of a certain substance refers to an amount of thesubstance that is sufficient to effectuate a desired result. Forinstance, an effective amount of a composition comprising a peptide ofthis invention that is intended to induce an anti-PAP immunity is anamount sufficient to achieve the goal of inducing the immunity whenadministered to a subject. The effect to be achieved may include theprevention, correction, or inhibition of progression of the symptoms ofa disease/condition and related complications to any detectable extent.The exact quantity of an “effective amount” will depend on the purposeof the administration, and can be ascertainable by one skilled in theart using known techniques (see, e.g., Lieberman, Pharmaceutical DosageForms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

A “physiologically acceptable excipient” is an inert ingredient used inthe formulation of a composition of this invention, which contains theactive ingredient(s) of a promiscuous PAP peptide or a fusion peptidecomprising the PAP peptide and a heterologous polypeptide and issuitable for use, e.g., by injection into a patient in need thereof.This inert ingredient may be a substance that, when included in acomposition of this invention, provides a desired pH, consistency,color, smell, or flavor of the composition.

As used herein, the term “T cell immune response” refers to activationof antigen specific T cells as measured by proliferation or expressionof molecules on the cell surface or secretion of proteins such ascytokines.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present inventors have identified novel promiscuous T cell epitopesfrom the human prostatic acid phosphatase (PAP) protein. These peptideepitopes demonstrate remarkable HLA promiscuity as they can be presentedin the context of at least 9 different HLA-DR serological families andat least 15 different HLA-DRβ1 alleles. The presentation of theseepitopes by such a wide range of HLA-DRβ1 alleles makes the epitopesextremely valuable as universal CD4 T helper cell epitopes inpreparation of vaccines or immunotherapies for the treatment of prostatecancer overexpressing the PAP protein in the general human population.

II. Chemical Synthesis of Peptides

The peptides of the present invention, particular those of relativelyshort length (e.g., no more than 50-100 amino acids), may be synthesizedchemically using conventional peptide synthesis or other protocols wellknown in the art.

Peptides may be synthesized by solid-phase peptide synthesis methodsusing procedures similar to those described by Merrifield et al., J. Am.Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-PhasePeptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Grossand Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980);and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem.Co., Rockford, Ill. (1984). During synthesis, N-α-protected amino acidshaving protected side chains are added stepwise to a growing polypeptidechain linked by its C-terminal and to a solid support, i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc, which is acid labile, and Fmoc,which is base labile.

Materials suitable for use as the solid support are well known to thoseof skill in the art and include, but are not limited to, the following:halomethyl resins, such as chloromethyl resin or bromomethyl resin;hydroxymethyl resins; phenol resins, such as4-(α-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin;tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins arecommercially available and their methods of preparation are known bythose of ordinary skill in the art.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe solid support. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arethen cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, e.g., Athertonet al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press(1989), and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed.,Springer-Verlag (1993)).

III. Recombinant Production of Peptides

A. General Recombinant Technology

Basic texts disclosing general methods and techniques in the field ofrecombinant genetics include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Ausubel et al., eds.,Current Protocols in Molecular Biology (1994).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett.22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange HPLC asdescribed in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

Recombinant production is an effective means to obtain peptides of thisinvention, particularly those of relatively large molecular weight, forexample, a fusion peptide of a promiscuous PAP epitope and a GM-CSF. Thesequence of a polynucleotide encoding a peptide of this invention, andsynthetic oligonucleotides can be verified after cloning or subcloningusing, e.g., the chain termination method for sequencing double-strandedtemplates of Wallace et al., Gene 16: 21-26 (1981).

B. Construction of an Expression Cassette

Obtaining a Polynucleotide Sequence Encoding a Peptide of the Invention

A polynucleotide sequence encoding a peptide of this invention can beobtained by chemical synthesis, or can be purchased from a commercialsupplier, which may then be further manipulated using standardtechniques of molecular cloning.

Modification of Nucleic Acids for Preferred Codon Usage in a HostOrganism

The polynucleotide sequence encoding a peptide of this invention can beoptionally altered to coincide with the preferred codon usage of aparticular host. For example, the preferred codon usage of one strain ofbacterial cells can be used to derive a polynucleotide that encodes apeptide of the invention and includes the codons favored by this strain.The frequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell (e.g., calculation service isavailable from web site of the Kazusa DNA Research Institute, Japan).This analysis is preferably limited to genes that are highly expressedby the host cell.

At the completion of modification, the coding sequences are verified bysequencing and are then subcloned into an appropriate expression vectorfor recombinant production of the peptides of this invention.

Following verification of the coding sequence, the peptide of thepresent invention can be produced using routine techniques in the fieldof recombinant genetics.

C. Expression Systems

To obtain high level expression of a nucleic acid encoding a peptide ofthe present invention, one typically subclones a polynucleotide encodingthe peptide into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator and aribosome binding site for translational initiation. Suitable bacterialpromoters are well known in the art and described, e.g., in Sambrook andRussell, supra, and Ausubel et al., supra. Bacterial expression systemsfor expressing a peptide of this invention are available in, e.g., E.coli, Bacillus sp., Salmonella, and Caulobacter. Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available. In one embodiment, theeukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes atranscription unit or expression cassette that contains all theadditional elements required for the expression of a peptide of thisinvention in host cells. A typical expression cassette thus contains apromoter operably linked to the polynucleotide sequence encoding thepeptide and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding the peptide is typically linked to acleavable signal peptide sequence to promote secretion of the peptide bythe transformed cell. Such signal peptides include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene (e.g., encoding theheterologous polypeptide), introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as abaculovirus vector in insect cells, with a polynucleotide sequenceencoding the peptide of this invention under the direction of thepolyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary. Similar toantibiotic resistance selection markers, metabolic selection markersbased on known metabolic pathways may also be used as a means forselecting transformed host cells.

When periplasmic expression of a recombinant protein (e.g., a peptide ofthe present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

C. Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast, insect, or plant cell lines that express large quantities of apeptide of this invention, which are then purified using standardtechniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)). Transformation of eukaryotic andprokaryotic cells are performed according to standard techniques (see,e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss,Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe peptide of this invention.

D. Detection of Recombinant Expression of a Peptide in Host Cells

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the peptide of this invention. The cells are then screened for theexpression of the recombinant peptide, which is subsequently recoveredfrom the culture using standard techniques (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a peptide of this invention intransfected cells can also be detected by PCR or RT-PCR usingsequence-specific primers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with apeptide of the present invention, particularly one containing asufficiently large heterolougs polypeptide (e.g., Harlow and Lane,Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988;Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniquesrequire antibody preparation by selecting antibodies with highspecificity against the peptide or an antigenic portion thereof. Themethods of raising polyclonal and monoclonal antibodies are wellestablished and their descriptions can be found in the literature, see,e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976).

IV. Purification of Peptides

A. Purification of Chemically Synthesized Peptides

Purification of synthetic peptides is accomplished using various methodsof chromatography, such as reverse phase HPLC, gel permeation, ionexchange, size exclusion, affinity, partition, or countercurrentdistribution. The choices of appropriate matrices and buffers are wellknown in the art.

B. Purification of Recombinantly Produced Peptides

1. Purification of Peptides from Bacterial Inclusion Bodies

When a peptide of the present invention is produced recombinantly bytransformed bacteria in large amounts, typically after promoterinduction, although expression can be constitutive, the peptides mayform insoluble aggregates. There are several protocols that are suitablefor purification of protein inclusion bodies. For example, purificationof aggregate proteins (hereinafter referred to as inclusion bodies)typically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, anon-ionic detergent. The cell suspension can be ground using a Polytrongrinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cellscan be sonicated on ice. Alternate methods of lysing bacteria aredescribed in Ausubel et al. and Sambrook and Russell, both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, may be inappropriate foruse in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of the immunologically and/orbiologically active protein of interest. After solubilization, theprotein can be separated from other bacterial proteins by standardseparation techniques. For further description of purifying recombinantpolypeptides from bacterial inclusion body, see, e.g., Patra et al.,Protein Expression and Purification 18:182-190 (2000).

Alternatively, it is possible to purify recombinant polypeptides, e.g.,a peptide of this invention, from bacterial periplasm. Where therecombinant polypeptide is exported into the periplasm of the bacteria,the periplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to those of skill in the art(see e.g., Ausubel et al., supra). To isolate recombinant peptides fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant peptides present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

2. Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., a peptide of the presentinvention, is expressed in host cells in a soluble form, itspurification can follow the standard protein purification proceduredescribed below. This standard purification procedure is also suitablefor purifying peptides obtained from chemical synthesis.

i. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest, e.g., a peptide of the presentinvention. The preferred salt is ammonium sulfate. Ammonium sulfateprecipitates proteins by effectively reducing the amount of water in theprotein mixture. Proteins then precipitate on the basis of theirsolubility. The more hydrophobic a protein is, the more likely it is toprecipitate at lower ammonium sulfate concentrations. A typical protocolis to add saturated ammonium sulfate to a protein solution so that theresultant ammonium sulfate concentration is between 20-30%. This willprecipitate the most hydrophobic proteins. The precipitate is discarded(unless the protein of interest is hydrophobic) and ammonium sulfate isadded to the supernatant to a concentration known to precipitate theprotein of interest. The precipitate is then solubilized in buffer andthe excess salt removed if necessary, through either dialysis ordiafiltration. Other methods that rely on solubility of proteins, suchas cold ethanol precipitation, are well known to those of skill in theart and can be used to fractionate complex protein mixtures.

ii. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of a protein of interest, e.g., a peptide of the presentinvention. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the peptide of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

iii. Column Chromatography

A protein of interest (such as a peptide of the present invention) canalso be separated from other proteins on the basis of its size, netsurface charge, hydrophobicity, or affinity for ligands. In addition,antibodies raised against a peptide of this invention can be conjugatedto column matrices and the peptide immunopurified. All of these methodsare well known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

C. Confirmation of Peptide Sequence

The amino acid sequence of a peptide of this invention can be confirmedby a number of well established methods. For example, the conventionalmethod of Edman degradation can be used to determine the amino acidsequence of a peptide. Several variations of sequencing methods based onEdman degradation, including microsequencing, and methods based on massspectrometry are also frequently used for this purpose.

D. Modification of Peptides

The peptides of the present invention can be modified to achieve moredesirable properties. The design of chemically modified peptides andpeptide mimics that are resistant to degradation by proteolytic enzymesor have improved solubility or binding ability is well known.

Modified amino acids or chemical derivatives of a promiscuous PAPpeptide or fusion peptides of this invention may contain additionalchemical moieties of modified amino acids not normally a part of the PAPprotein. Covalent modifications of the peptides are within the scope ofthe present invention. Such modifications may be introduced into apeptide by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The following examples of chemicalderivatives are provided by way of illustration and not by way oflimitation.

The design of peptide mimics which are resistant to degradation byproteolytic enzymes is known to those skilled in the art. See e.g.,Sawyer, Structure-Based Drug Design, P. Verapandia, Ed., N.Y. (1997);U.S. Pat. Nos. 5,552,534 and 5,550,251. Both peptide backbone and sidechain modifications may be used in designing secondary structuremimicry. Possible modifications include substitution of D-amino acids,N^(α)-Me-amino acids, C_(α)-Me-amino acids, and dehydroamino acids. Tothis date, a variety of secondary structure mimetics have been designedand incorporated in peptides or peptidomimetics.

Other modifications include substitution of a natural amino acid with anunnatural hydroxylated amino acid, substitution of the carboxy groups inacidic amino acids with nitrile derivatives, substitution of thehydroxyl groups in basic amino acids with alkyl groups, or substitutionof methionine with methionine sulfoxide. In addition, an amino acid of apromiscuous PAP peptide or a fusion peptide of this invention can bereplaced by the same amino acid but of the opposite chirality, i.e., anaturally-occurring L-amino acid may be replaced by its D-configuration.

V. Fusing a PAP Epitope with a Heterologous Polypeptide

In one aspect of this invention, a peptide corresponding to apromiscuous PAP epitope is attached to a heterologous polypeptide via acovalent bond to form a fusion peptide, such that the ability of the PAPepitope to induce a T cell response is enhanced. Frequently, thiscovalent bond is a peptide bond and the PAP epitope and the heterologouspolypeptide form a new polypeptide. This peptide bond may be a directpeptide bond between the PAP epitope and the heterologous polypeptide,or it may be an indirect peptide bond provided by way of a peptidelinker between the PAP epitope and the heterologous polypeptide.

Other covalent bonds are also suitable for the purpose of fusing the PAPpeptide with the heterologous polypeptide. For instance, a functionalgroup (such as a non-terminal amine group, a non-terminal carboxylicacid group, a hydroxyl group, and a sulfhydryl group) of one peptide mayeasily react with a functional group of the other peptide and establisha covalent bond, other than a peptide bond, that conjugates the twopeptides. A covalent connection between a peptide of a promiscuous PAPepitope and a heterologous polypeptide can also be provided by way of alinker molecule with suitable functional group(s). Such a linkermolecule can be a peptide linker or a non-peptide linker. A linker maybe derivatized to expose or to attach additional reactive functionalgroups prior to conjugation. The derivatization may involve attachmentof any of a number of molecules such as those available from PierceChemical Company, Rockford, Ill.

VI. Functional Assays

A promiscuous PAP epitope of this invention (or a fusion peptidecomprising the PAP peptide and a heterologous polypeptide) is useful forits capability to induce a T cell immune response specific to a PAPprotein, when the epitope is presented by an antigen-presenting cellthat may have one of at least 10 different HLA-DR alleles, morepreferably at least 12, 13, 14, or 15 different HLA-DR alleles. Variousfunctional assays can be used to confirm the ability of a promiscuousPAP epitope to induce such a PAP-specific T cell immune response in apromiscuous manner with regard to antigen presenting cells of differentHLA-DR alleles, including proliferation assay and flow cytometry assaysdetecting the binding between a T cell receptor and a peptide epitope orthe production of cytokines by T cells.

The functional assay system used in the Examples of this application isparticularly suitable for this purpose. Briefly, a panel of at least 10,preferably at least 12, 13, 14, 15 or more antigen presenting celllines, each homozygous for a different HLA-DR allele, is employed topresent a PAP-derived peptide to a CD4+ T cell clone (e.g., clonePAPc66) that is specifically responsive to the PAP protein (e.g., byproduction of cytokines such as IFNγ or granzyme B). The peptidecorresponding to residues 257-271 of the human PAP protein (i.e., SEQ IDNO: 1) is used as a positive control. An irrelevant PAP-derived peptide,no peptide, and each antigen presenting cell line alone are used asnegative controls for the assays. The assays are set up in multi-welledcell culture plates in appropriate medium with antigen presenting cellsand CD4⁺ T cells in each well. Peptides are diluted to a suitableconcentration and added to each well. Following incubation of anappropriate time period, supernatants are collected from the wells andanalyzed for cytokine production, which can be measured by ELISA basedon absorbance at 492 nm. Typically, the effect of a PAP class IIpromiscuous eptiope of this invention in inducing a PAP-specific CD4⁺ Tcell response is at least 25% of the effect of PAP epitope 257-271(which has the amino acid sequence set forth in SEQ ID NO: 1) under thesame assay conditions, e.g., at the same molar concentration andpresented by antigen-presenting cells of the same, individual HLA-DRallele. More preferably such effect is at least 30%, 40%, 50%, 60%, 70%,80% or higher of that shown by PAP epitope 257-271 under the sameconditions.

VII. Compositions and Administration

The present invention also provides compositions comprising an effectiveamount of (1) a promiscuous PAP peptide; or (2) a fusion peptidecomprising a PAP peptide and a heterologous polypeptide; or (3) anantigen presenting cell (APC) with the peptide of (1) or (2) forming acomplex with an MHC molecule on the cell surface for inducing a T cellimmune response specific against a PAP protein in both prophylactic andtherapeutic applications. Pharmaceutical compositions of the inventionare suitable for use in a variety of drug delivery systems. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). For a brief review of methods for drug delivery, see,Langer, Science 249: 1527-1533 (1990).

Antigen presenting cells (APCs) can be generated for peptide loading bya variety of methods. The starting raw material is peripheral blood or aleukapheresis with or without mobilization. APCs can be isolated bymultiple methods, e.g., buoyant density centrifugation, elutriation,magnetic beads and plastic adherence used alone or in combination. Afterisolation, APCs are cultured for 1-14 days with or without the presenceof cytokines, growth factors, activation agents and maturation agents.APCs are loaded with peptide by addition of peptide to the culture inconcentrations from 1 μg to 1 mg/mL for 6-48 hrs. APCs are thenharvested, washed and resuspended in a suitable formulation forinfusion. APCs can be delivered fresh or can be kept in frozen storagefor delivery at a later time.

The pharmaceutical compositions of the present invention can beadministered by various routes, e.g., subcutaneous, intradermal,transdermal, intramuscular, intravenous, or intraperitoneal. Thepreferred routes of administering the pharmaceutical compositions aresubcutaneous or intradermal at biweekly doses of about 1 μg-10 mg,preferably 50 μg-1 mg, of a peptide of this invention for a 70 kg adulthuman. The appropriate dose may be administered in weekly, biweekly, ormonthly intervals.

Peptide pulsed APCs can be administered by various routes, e.g.,subcutaneous, intradermal, intravenous or intraperitoneal. The peptidepulsed APCs are delivered in weekly, biweekly, or monthly intervals atdoses of 1 million to 10 billion cells.

For preparing pharmaceutical compositions containing a peptide of thepresent invention, inert and pharmaceutically acceptable excipients orcarriers are used. Liquid pharmaceutical compositions include, forexample, solutions, suspensions, and emulsions suitable for intradermal,subcutaneous, parenteral, or intravenous administration. Sterile watersolutions of the active component (e.g., a promiscuous PAP peptide orfusion peptide) or sterile solutions of the active component in solventscomprising water, buffered water, saline, PBS, ethanol, or propyleneglycol are examples of liquid compositions suitable for parenteraladministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, detergents, and the like.

Sterile solutions can be prepared by dissolving the active component(e.g., a promiscuous PAP peptide or fusion peptide) in the desiredsolvent system, and then passing the resulting solution through amembrane filter to sterilize it or, alternatively, by dissolving thesterile compound in a previously sterilized solvent under sterileconditions. The resulting aqueous solutions may be packaged for use asis, or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of thepreparations typically will be between 3 and 11, more preferably from 5to 9, and most preferably from 7 to 8.

The pharmaceutical compositions containing a PAP peptide or fusionpeptide can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a patient already suffering from a condition that may be exacerbatedby the proliferation of tumor cells overexpression the PAP protein in anamount sufficient to prevent, cure, reverse, or at least partially slowor arrest the symptoms of the condition and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.” Amounts effective for this use will depend on the severity of thedisease or condition and the weight and general state of the patient,but generally range from about 1 μg to about 10 mg of the PAP peptide orfusion peptide biweekly for a 70 kg patient, with dosages of from about50 μg to about 1 mg of the peptide biweekly for a 70 kg patient beingmore commonly used. The appropriate dose may be administered in weekly,biweekly, or monthly intervals.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of a PAP peptide or fusion peptide of this inventionsufficient to effectively inhibit PAP-expressing tumor cellproliferation in the patient for therapeutic purposes.

VIII. Method for Detecting T Cell Response Specific to PAP

The present invention further provides a method for detecting whether aT cell immune response specific to a PAP protein is present in apatient. This method includes the following steps: first, lymphocytesincluding at least a T cell and an antigen-presenting cell are obtainedfrom a patient. Suitable samples that yield such lymphocytes includeblood, tumor infiltrate, and lymph nodes or lymphatic fluids. Second,the T cell and antigen-presenting cells are exposed to a promiscuous PAPpeptide (or a fusion peptide comprising the PAP peptide and aheterologous peptide) of this invention under conditions that wouldallow proper presentation of a T cell epitope by the antigen-presentingcell to the T cell. Third, signs of a T cell response is measured invitro by means well known in the art such as ELISPOT, proliferationassay, or flow cytometry. When a T cell response is detected by any ofthese methods, it can be concluded that there exists a T cell immuneresponse specific to a PAP protein in the patient.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

Example 1 Materials and Methods

Recombinant Proteins and Synthetic Peptides. PA2024 is a proprietaryrecombinant fusion protein containing PAP and GM-CSF sequencesmanufactured by Dendreon Corporation for the investigational cellularimmunotherapy sipuleucel-T. PA2024 is expressed in a baculovirus system.CHOPA2024 and hPAPGM are sequence identical to PA2024 but expressed inmammalian systems using Chinese Hampster Ovary cells (CHO) and 293 Ebnarespectively. iPAP is a recombinant protein produced in a baculovirusexpression system and CHOPAP is a recombinant protein produced inmammalian cells, both produced and purified by Dendreon Corporation. Fordefining PAP specific immune responses in vitro, 94 peptides weregenerated from the PAP protein sequence. These peptides were 15 aminoacids in length, overlapping by 11 amino acids (Genemed Synthesis, SouthSan Francisco, Calif.). All PAP peptides were sequenced and determinedto be >95% pure by analytical HPLC and mass spectroscopy.

Subject and Healthy Donor Sample Collection. All subject and healthydonor specimens were collected according to investigator-sponsoredprotocols approved by the appropriate Investigational Review Board.Following informed consent, whole blood samples were collected byvenipuncture into heparinized vacutainer tubes or syringes and preparedfor transport and/or processing. After receipt of blood samples by thelaboratory, peripheral blood mononuclear cells (PBMC) were collectedunder sterile conditions by density gradient centrifugation and preparedfor use in specified assays.

Cell Lines. HLA-DRβ1 EBV-LcL lines were purchased from the EuropeanCollection of Cell Cultures originating from the 12^(th) InternationalHistocompatibility Workshop (IHW) held in Strasbourg, France. HLA-DREBV-transformed lymphoblastoid cells were homozygous for the HLA-DRβ1allele (Table I). EBV-LcL were also generated from various subject PBMCusing supernatant from the B95-8 cell line (ATCC, Manassas, Va.) for theexpansion and testing of autologous T cell clones.

In Vitro Generation of PAP specific T cell clones. PBMC from a subjectreceiving sipuleucel-T were stimulated in a T-25 tissue culture flaskwith 10 μg/mL of CHOPA2024 overnight in RPMI 1640 with 2 mM L-glutamine,50 U/mL Penicillin, 50/g/mL Streptomycin and 20 mM HEPES buffer with 10%Human AB serum (Gemini BioProducts, Calabasas, Calif.) (cRPMI+10% HS).The following day, IFNγ secreting cells were isolated from the PBMCculture using the IFNγ Secretion Assay Cell Enrichment and Detection kit(Miltenyi Biotech, Auburn, Calif.). The IFNγ enriched population wasplated for cloning in 96 well round bottom plates at approximately 1-50cells/well with 10 U/mL recombinant human IL-2 (Invitrogen). Non-IFNγsecreting cells were irradiated (3000 rads) and used as feeder cells.Plates were incubated for seven days at 37° C. with 5% CO₂. On day 7 ofthe cloning, IFNγ secreting cells were non-specifically expanded in 96well plates as previously described (Yee et al., Proc. Natl. Acad. Sci,99: 16168-16173). Briefly, 100 μL of cRPMI+10% HS media with 25 U/mLrecombinant human IL-2 and 10 ng/mL anti-human CD3 antibody (BDPharmingen, San Diego, Calif.) was added to each well with 1×10⁴/wellirradiated autologous EBV-LcLs and 1×10⁵/well irradiated allogeneicPBMC. Plates were incubated for 14 days at 37° C. with 5% CO₂ and thenvisually inspected for positive growth. Growth positive clones weretransferred into 24 well plates and expanded using rIL-2, anti-CD3 andaccessory cells as above, adjusting cell numbers for the increasedvolume.

Antigen Specificity of T cell clones. All assays were set up intriplicate in 96-well round bottom plates with antigen or peptide incRPMI 1640+10% FBS (Fetal Bovine Serum (Invitrogen, Carlsbad, Calif.)),T cell clones at 1×10⁵ cells/well and antigen presenting cells at 2×10⁵cells/well. Autologous EBV-LcL were used for antigen presenting cells inspecificity assays and assays conducted to show peptide promiscuityusing the HLA-DRB1 EBV-LcLs. All assays were incubated for 48 hours at37° C. with 5% CO₂. After 48 hours, supernatants were harvested to assayfor cytokine production. MHC Class II blocking assays were set up asabove with autologous EBV-LcL and the addition of the followingantibodies (25 μg/mL-0.1 μg/mL) to the assay: anti-HLA-DR mAb (DendreonCorporation and Research Diagnostics, Inc. Flanders, N.J.), HLA-DQ mAb1a3 and HLA-DP mAb B7/21 (Leinco Technologies, St. Louis, Miss.).Anti-HLA-A2 mAb (HB-82, ATCC, Manassas, Va.) was used as a control inblocking assays.

IFNγ and Granzyme B ELISA. IFN□ production was measured using anti-humanIFNγ antibody pairs for ELISA (BD Pharmingen, San Diego, Calif.).Briefly, Immulon 4 plates (Thermo Labsystems/VWR, Brisbane, Calif.) werecoated overnight with 100 μL of anti-human IFNγ antibody at 3 μg/mL.Plates were blocked with 4% Bovine Serum Albumin (BSA) (Sigma, St.Louis, Mo.) in PBS (Invitrogen) for 2 hours at 37° C. Plates were washedwith PBS+0.05% Tween 20 and 100 μL of supernatant samples from theantigen specific stimulation was added to wells and incubated at roomtemperature for 1.25-2 hours. Plates were washed and biotinylatedanti-human IFNγ antibody was diluted in 1% BSA in PBS (1 μg/mL) andadded to plates in 100 μL for 1 hour at room temperature. After washing,Strep-Avidin HRP (BD Pharmingen) was diluted 1:1000 in PBST and added towells for 30 minutes at room temperature. Plates were washed incubatedwith Sigma Fast OPD for 15 minutes in the dark. 2M HCl was added to stopthe reaction and plates were read for absorbance at 492 nm on aspectrophotometer. Granzyme B ELISA kit (Cat. No. 3485-1H-20, Mabtech,Nacka Strand, Sweden) was used to measure granzyme B production. Coatingantibody (GB10), biotinylated detection antibody (GB11) andStrep-Avidin-BRP were used according to manufacturer's recommendationsunder the protocol described above.

Results

The experimental results shown in FIGS. 1-3 demonstrate that peptidePAP₂₅₇₋₂₇₁ is a naturally processed, MHC class II-restricted promiscuousepitope of prostatic acid phosphatase (PAP). This peptide has the aminoacid sequence as shown in SEQ ID NO: 1 (RLQGGVLVNEILNHM), is capable ofinducing T-cell activation when presented by lymphoblastoid cell linesrepresenting 9 different HLA-DR serological families (β chain; DR1, DR4,DR7, DR8, DR11, DR12, DR13, DR14, and DR15) having over 15 differentHLA-DRβ1 alleles.

The CD4+ T cell clone, PAPc66, was isolated from a subject participatingin an ongoing phase 3 clinical trial for the treatment of prostatecancer using sipuleucel-T, which is an investigational autologous activecellular immunotherapy product designed to stimulate T-cell immuneresponses against human PAP. After an overnight stimulation of subjectperipheral blood mononuclear cells with a fusion protein of PAP andhuman granulocyte-macrophage colony-stimulating factor (PA2024), PAPc66was isolated using IFNγ secretion as a marker for selection. Theclinical trial subject from whom this clone was isolated was typed asHLA-DRB1*0404, 1501. This clone has also been shown to produce IFNγ andgranzyme B upon activation with appropriately presented PAP₂₅₇₋₂₇₁.

This novel promiscuous epitope provides a tool for investigating thepotential immune mechanisms occurring as a consequence of treatment withPAP-specific, active cellular immunotherapy. Additionally, this peptidecontributes to more universal targeting strategies for enhancing futurecancer immunotherapies.

TABLE I HLA-defined EBV-LCL cell lines. HLA-DRB1* DR serological Cellline allele family name 0101 DR1 KAS116 0102 DR1 PMG075 0103 DR103TER-ND 1503 DR15 AMAI 160201 DR16 RML 0301 DR17 VAVY 0302 DR18 RSH 0401DR4 BM14 0402 DR4 YAR 040301 DR4 SSTO 040501 DR4 LKT3 1101 DR11 BM211102 DR11 BM15 1103 DR11 TISI 1104 DR11 BOB 110401 DR11 FPAF 1201 DR12BM16 1301 DR13 OMW 1302 DR13 EMJ 1401 DR14 EK 1402 DR14 AMALA 0701 DR7BER 080101 DR8 BM9 080201 DR8 SPL 0901 DR9 T7526Cell lines were obtained from the ECACC European Collection of CellCultures and are listed in the IMGT/HLA cell directory (websiteebi.ac.uk/imgt/hla/cell query.html).

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in the entirety for all purposes.

1. An isolated peptide consisting of 15 to 18 amino acids and comprisingthe amino acid sequence of SEQ ID NO:1.
 2. A fusion peptide consistingof the isolated peptide of claim 1 fused to a heterologous peptide. 3.The fusion peptide of claim 2, wherein the isolated peptide of claim 1is fused to the heterologous peptide via a peptide bond.
 4. The isolatedpeptide of claim 1, which induces a T cell immune response specific tohuman prostatic acid phosphatase (PAP) protein when presented by anantigen-presenting cell in the context of at least 10 different HLA-DRalleles.
 5. The isolated peptide of claim 4, which induces a T cellimmune response specific to a PAP protein when presented by anantigen-presenting cell in the context of at least 15 different HLA-DRalleles.
 6. The isolated peptide of claim 4, wherein the HLA-DR allelesare selected from the group consisting of 0101, 0102, 0103, 1503,160201, 0301, 0302, 0401, 0402, 040301, 040501, 1101, 1102, 1103, 1104,110401, 1201, 1301, 1302, 1401, 1402, 0701, 080101, 080201, and
 0901. 7.The isolated peptide of claim 1, which consists of the amino acidsequence of SEQ ID NO:1.
 8. The fusion peptide of claim 2, wherein theisolated peptide consists of the amino acid sequence of SEQ ID NO:1. 9.The fusion peptide of claim 2, wherein the heterologous peptide is agranulocyte-macrophage colony-stimulating factor (GM-CSF).
 10. Acomposition comprising the isolated peptide of claim 1 and aphysiologically acceptable excipient.
 11. A composition comprising thefusion peptide of claim 2 and a physiologically acceptable excipient.12. The composition of claim 10, wherein the isolated peptide consistsof the amino acid sequence of SEQ ID NO:1.
 13. The composition of claim11, wherein the isolated peptide consists of the amino acid sequence ofSEQ ID NO:1.
 14. The composition of claim 11, wherein the heterologouspeptide is a granulocyte-macrophage colony-stimulating factor (GM-CSF).15. The composition of claim 10, further comprising anantigen-presenting cell with the isolated peptide forming a complex witha major histocompatibility complex (MHC) molecule on the surface of thecell.
 16. A method for detecting in a patient a T cell immune responsespecific to human prostatic acid phosphatase (PAP) protein, the methodcomprising the steps of (a) obtaining an antigen-presenting cell and a Tcell from the patient; (b) contacting the antigen-presenting cell andthe T cell with the isolated peptide of claim 1; and (c) detecting a Tcell response, wherein the detection of a T cell response indicates thepresence of a T cell immune response specific to the PAP protein in thepatient.
 17. The method of claim 16, wherein step (c) is performed byELISPOT, proliferation assay, or flow cytometry.
 18. The method of claim16, wherein the isolated peptide consists of the amino acid sequence ofSEQ ID NO:1.